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Background

Recent years, though agricultural pesticides have brought huge benefits to the production of crops worldwide, it also posed significant threat to human health. According to the report from Food and Agriculture Organization of the United Nations (FAO), humans have used an incredible amount of pesticides of 3.70 million tons globally in the year 2022.
Figure 1 pesticide use in 2022
Figure 1. Pesticide use in 2022
Among all, China has almost become the country with the largest amount of pesticide application. This results in the presence of a large amount of pesticide residues in the fruits and vegetables that are sold and consumed by people. The pesticides that are widely adopted often contain organic phosphates (OCPs) (Mulla et al., 2020), which can lead to serious health problems in human bodies (James et al., 1993). OCPs can cause neurological disorders, such as essential tremor (Louis et al., 2006), and PCBs can lead to neurological diseases, liver problems, and even pass on to children (Reggiani & Bruppacher, 1985). Though some might argue that the effect of pesticide residue on human is not obvious in the short time, what truly worrying is their chronic health effects, as illustrated in the graph below.
Figure 2 acute & chronic health effects with pesticide use
Figure 2. Acute & chronic health effects with pesticide use
Furthermore, the current regulation of the use of organophosphorus pesticides in China is difficult to achieve comprehensively. The main limitation is the lack of rapid detection methods.With the country's strengthened control over food security and people’s growing pursuit for a healthy lifestyle, new demands were raised regarding pesticide residue detection.

Current Solution

The current major solutions of pesticide residues detection can be divided into 2 types—precise, quantitative detection method (include chromatography) and rapid detection method (include the enzymatic inhibition method and the enzyme-linked immunosorbent assay).

Although there were several different methods of detecting pesticide residues presented (Xu et al., n.d.), shortcomings were shown when using those approaches. The liquid chromatography-mass spectrometry (LC-MS) method is a common way to detect the residues. It has a high sensitivity, but the cost is far higher to apply in production use (Lim & Lord, 2002); traditional acetylcholinesterase (AChE) detection is more effective and cheaper in cost (Handali & Webb, 2023), but it relies on the enzyme, which is unstable (Tsounidi et al., 2023).
Method Advantages Limitations
Chromatography 1. Precise quantitative analysis
2. Good stability 		1. Limited types of detectable pesticides
2. Complicated operation steps
3. Hard for mass testing
4. High machine maintaining cost
Enzyme-linked Immunosorbent Assay (ELISA) 1. High specificity, avoiding interference
2. Easy to detect, reaching mass testing 		1. Poor stability, prone to loss activity
2. High store requirement
3. Not suitable for all kinds of pesticide
Enzyme Inhibition Method 1. Convenient operation
2. Low cost, suitable for daily testing 		1. Unstable enzyme activity, hard to store
2. Qualitative but not quantitative
Considering that the Enzyme Inhibition Method is more convenient and conducive to rapid detection as well as practical use in daily life, and given that the current method is unstable and inefficient and urgently needs to be updated, we decided to optimize the Enzyme Inhibition Method in rapid detection of pesticide residues using synthetic biology.

Design Principle

As exemplified in its name, the core of Enzyme Inhibition Method is the adoption of enzyme Acetylecholinesterase (AChE). This is a kind of rapid pesticide residue detection method that can be integrated on paper devices such as test strips (Fuyal & Giri, 2020).
The principle of Enzyme Inhibition Method is shown in the graph below. AChE catalyzes the hydrolysis of Indoxyl Acetate (a common substrate that places on the test strip) into indophenol and acetic acid, producing a measurable color change. However, organophosphate and carbamate pesticides inhibit AChE activity by binding to its active site, preventing substrate hydrolysis. On the test strip, this inhibition leads to a reduction in color intensity proportional to the pesticide concentration.
Figure 3 Principle of Enzyme Inhibition Method
Figure 3. Principle of Enzyme Inhibition Method

Our Solution

In our project, we aim to address several drawbacks in the existing pesticide residue including the low sensitivity and stability. Aiming for this goal, our team came up with the solution Pestacheck. We integrated techniques from synthetic biology, nanotechnology and graphical identification technology into the following steps:
  1. Target Selection and Mutagenesis
    We began by analyzing the structural characteristics of our target enzyme AChE. To investigate key amino acid residues that determine enzyme activity and stability, we performed Alanine scanning mutagenesis.

    In molecular biology, alanine scanning is a site-directed mutagenesis technique used to determine the contribution of a specific residue to the stability or function of a given protein. The alanine scanning method exploits the fact that most standard amino acids can be replaced by alanine through point mutations, while the secondary structure of the mutated protein remains unchanged because alanine can simulate the secondary structure preferences of most encoded amino acids or standard amino acids.

    Through this method, we introduces several point mutations for the wild-type AChE. This allowed us to explore how specific sites contribute to the enzyme’s catalytic function and optimize its performance for detection purposes.
    Figure 4. Alanine Scanning
    Figure 4. The mutation sites
    Figure 5. The mutation sites
  2. Comparing Expression Systems
    Initially, AChE and its mutants were expressed in the E. coli system. However, the results revealed low solubility and enzyme activity, likely due to improper protein folding and inclusion body formation in the prokaryotic environment. After conversation with field experts and self-exploration, we were inspired by the 2019 iGEM Thessaly team with their incorporation of cell-free synthesis, which provides "unprecedented freedom of design in an open environment than cell system" (Liu, 2017). Therefore, we adopted a Cell-Free Protein Synthesis (CFPS) system, which enabled efficient, controllable, and functional protein production. CFPS offers multiple advantages, including flexibility, safety, and rapid prototyping for synthetic biology applications (Khambhati et al., 2019). Finally, we obtained soluble proteins through CFPS, and through functional and stability experiments, we discovered the advantages of D203A. Therefore, we can proceed with the design of subsequent test strips.
    Figure 6. Cell-Free expression system
    Figure 6. Cell-Free expression system
  3. Enhancing Enzyme Stability and Sensitivity
    To further improve enzyme stability and prepare for the development of test strips, we immobilized the mutant enzymes onto gold nanoparticles (AuNPs). The AuNPs act as nanoscale scaffolds with adjustable size, modifiable surface chemistry, and a large surface area (Gai et al., 2023). This coupling process not only stabilized the enzyme structure but also enhanced the sensitivity of the detection signal for our designed detection system.
  4. Building a Smart Detection Platform
    We integrated the AChE–AuNP conjugate into a muti-layered colloidal gold test strip. The platform’s layered design ensures specificity and reliability in residue detection.
Figure 5. Pestacheck Solution Overview
Figure 7. Pestacheck Solution Overview
Product Description
Our synthetic biology solution Pestacheck contains several components. As a colloidal gold test strip, it contains several components:
  • Sample Pad: The point of application for the vegetable/fruit sample.
  • AChE-AuNP Conjugate: The key detection reagent, containing the enzyme immobilized with gold nanoparticles
  • Substrates (Indoxyl Acetate & Diazo Red RC): The substrate and dye that react to generate the visible color signal.
  • Absorbent Pad: Drives the capillary flow and wicks the excess fluid.
  • Plastic Backing: Provides structural integrity to the entire strip assembly.
During the detection process, the sample droplet to be tested is placed on the sample pad. Through capillary action, it moves along the test paper layer chromatography, re-dissolves and drives the AChE-AuNP together to migrate. If the sample does not contain pesticides, AChE will maintain its activity. During the flow process, it catalyzes the hydrolysis of the substrate indoxyl acetate to generate indoxyl，providing the blue signal. Conversely, if the sample contains pesticides, the pesticide will irreversibly inhibit the activity of AChE, preventing the subsequent catalytic and color-developing reactions from proceeding. As a result, no color band will appear in the detection area, and thus it will be determined as positive.
Figure 5 Pestacheck Platform
Figure 8. Pestacheck Platform

Conclusion

Through Pestacheck, we successfully combined synthetic biology, nanotechnology and digital analysis to enhance the accuracy, stability and operability of the current pesticide residue detection. By optimizing acetylcholinesterase (AChE) through site-directed mutagenesis, using the CFPS expression system and combining the enzyme with gold nanoparticles, we improved the performance and detection sensitivity of the enzyme. In summary, our system provides a practical, low-cost and user-friendly solution to ensure food safety and address the global challenge of pesticide pollution.

References

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